27 Jan 2023

While people who carry one hypofunctional variant in the TREM2 gene have a heightened risk for Alzheimer’s disease, those with two dysfunctional copies of this microglial receptor, or its partner, DAP12, face a more severe fate. They develop Nasu-Hakola disease, an autosomal-recessive condition that begins in adolescence and leads to early dementia and bone abnormalities. Mechanisms underlying this exceedingly rare disorder have been difficult to pin down. Now, scientists led by Marco Colonna at Washington University in St. Louis have conducted the first single-cell gene-expression study of brain samples from three people who died with the disease. In Nature Immunology on January 19, they report that microglia in the NHD brain had cranked up expression of genes involved in injury repair. Astrocytes engaged in damage control as well, while oligodendrocytes squelched myelination, and the signatures of pericytes and perivascular macrophages suggested the brain vasculature was damaged.

Ironically, the microglial shift toward tissue repair pathways could exacerbate damage in the brain, Colonna told Alzforum. Left unchecked, wound healing responses may counter other important functions of glial cells, he said. Strikingly, the study found scant overlap between these NHD signatures and those in a mouse model of NHD. The transcriptomic states also bore little resemblance to those found in heterozygous carriers of TREM2 variants associated with AD. “These risk variants may cause modest microglia dysfunction that facilitates plaque accumulation over an extended period of time in AD-susceptible individuals,” the authors wrote.

From its perch on myeloid cells throughout the body, including microglia in the CNS, osteoclasts in the bone, and macrophages in the liver, TREM2 senses lipids and other ligands within its microenvironment. Once stimulated, it signals via DAP12, also known as TYROBP, unleashing a cascade of tyrosine phosphorylation that promotes different transcriptional states. Though rare, homozygous mutations in TREM2 or DAP12 that block this signaling cascade cause NHD (Paloneva et al., 2001; Paloneva et al., 2002). Neuropathological hallmarks of the disease include brain atrophy in frontal regions, loss of myelin, axonal spheroids in the white matter, and gliosis. How loss of TREM2 signaling leads to this cerebral mayhem has been a mystery, since neither TREM2- nor DAP12-deficient mice develop the characteristic features of the disease, and human tissue samples are rare.

To gain insight into NHD pathogenesis, first author Yingyue Zhou and colleagues partnered with Akiyoshi Kakita at Niigata University and Masaki Takao at Mihara Memorial Hospital brain bank in Isesaki, Japan, who shared occipital cortex samples from three people with NHD and from 11 controls. The NHD cases were caused by homozygous loss-of-function mutations in DAP12. The researchers used single-nucleus RNA-Seq to profile the transcriptomes of more than 66,000 cells from the samples. Among the major cell types identified, microglia and perivascular macrophages were the sole expressors of DAP12. Astrocytes in the NHD brain samples had the most differentially expressed genes relative to controls, followed by oligodendrocytes, microglia, and vascular cells. Across these cell types, more genes were turned up than turned down.

In NHD microglia, the researchers found strong upregulation of three major regulatory proteins: RUNX1, which controls microglial proliferation in response to injury; STAT3, a transcription factor that influences all manner of tissue repair and anti-inflammatory genes; and TGF-β, a protein that helps maintain microglial homeostasis. These and other gene-expression changes indicated a unique injury-response signature, the authors contend. Notably, this profile was not observed in transcriptomic datasets from people with other neurodegenerative diseases, including AD, vascular dementia, and multiple sclerosis. However, the microglial NHD signature did align well with that of macrophages stimulated with IL-10, an anti-inflammatory cytokine.

While microglia in an anti-inflammatory, reparative mood might sound good for the brain, Colonna thinks it may limit the activation of other important defensive responses. “Unrestricted signaling of these pathways may dysregulate the ability of microglia to phagocytose apoptotic cells and myelin debris, and enhance profibrotic functions, ultimately resulting in neuron loss, demyelination and compensatory astrogliosis,” the authors wrote.

How did other cell types fare in the NHD brain? Astrocytes ramped up the activation marker GFAP, genes involved in tissue repair and wound healing, and genes that dictate reactive astrocytosis. Gene expression in vascular cells, including the endothelial cells that bolster vessel walls and the pericytes that wrap around them, were dramatically altered, and the vessels narrowed and thickened, suggesting a contracted and dysfunctional vasculature (see image below). Oligodendrocytes turned down myelin-making genes, in keeping with the demyelination seen in the disease. Finally, neurons turned down expression of genes involved in protein translation and synaptic transmission, reflecting a state of neurodegeneration. Overall, the transcriptomic findings aligned with the pathology observed in these NHD brain samples, which included astrogliosis, narrow and thick blood vessels, massive demyelination, and neurodegeneration.

Vascular Dysfunction. Compared to vessels in brain sections from controls (left), the brain blood vessels in people with NHD (right) were narrower and had thick walls. [Courtesy of Zhou et al., Nature Immunology, 2023.]

Colonna added that the findings also hint at a role for perivascular macrophages in the disease, which also express TREM2 and DAP12. The cells play a crucial role in maintaining blood vessels in the brain, so their malfunction could lead to vascular abnormalities and damage to white matter, which could in turn fire up tissue repair responses in astrocytes and microglia, Colonna said. The analysis was conducted on samples from people in end-stage disease, making it difficult to tease apart causative and responsive pathways.

Multi-pronged Pathogenesis. A defect in TREM2/DAP12 signaling promotes pathways driven  by STAT3, RUNX1, and TGFβ in microglia. This shift somehow promotes demyelination, which leads to accumulation of myelin and cellular debris, and tissue damage. This further activates microglia. DAP12 deficiency in perivascular macrophages (PVM) may also contribute to the disease cascade. [Courtesy of Zhou et al., Nature Immunology, 2023.]

The KD75 mouse model of NHD did not recapitulate these phenotypes (Tomasello et al., 2000). In these mice, which express a mutated form of DAP12 that thwarts its interaction with, and thus signaling through, TREM2, the researchers identified the same four transcriptional clusters of microglia as they did in wild-type mice. However, relative to wild-type mice, KD75 mice had markedly fewer disease-associated microglia. DAMs are driven by TREM2/DAP12 signaling and proliferate in models of amyloidosis. As opposed to the dramatically upregulated tissue repair response in people with NHD, KD75 microglia largely downregulated genes, including those involved in lipid metabolism and endolysosomal function. Effects on other cell types were modest, and included downregulation of activation genes in astrocytes, a subtle downregulation of myelin genes in oligodendrocytes, and virtually no effect in neurons. In keeping with these expression findings, KD75 mice exhibited little brain pathology.

What explains the stark differences between people and mice? There are many possible explanations, including species differences in TREM2 stimuli, or the presence of other receptors that might compensate for DAP12 loss in mice, said Colonna. He noted that the adolescent onset of NHD might offer a clue. Substantial remodeling of brain circuitry kicks into high gear during puberty, and restructuring of existing circuits is a part of that, Colonna said. Perhaps this “microdamage” typically engages DAP12 signaling, and without it, tissue repair runs amok in NHD. In mice, these same adolescent changes might not take place, he said.

Samuel Gandy, Michelle Ehrlich, and Jean-Vianney Haure-Mirande from the Icahn School of Medicine at Mount Sinai, New York, think that the mild brain pathology observed in the KD75 mice aligns with those of conventional DAP12 knockouts, however, they have found scant overlap between the transcriptomes of the two mouse lines. “In our opinion, the limited overlap … argues that the KD75 DAP12 mouse is not equivalent to a conventional DAP12 knockout,” they wrote. “It is unclear whether these differences are functionally important, but there is a very important similarity in that neither model recapitulates NHD.”

Cross-species differences in microglia might also explain model deficiencies. Oleg Butovsky of Brigham and Women’s Hospital in Boston suggested studying chimeric mice (Apr 2019 conference news; Apr 2019 news; Jan 2023 news). By repopulating mice with DAP12-deficient human microglia, researchers could find out whether the human microglia are sufficient to drive the disease. Chimeras could also help nail down other cells that contribute to the brain pathology of NHD.—Jessica Shugart

Comments

1. • Sam Gandy
     Icahn School of Medicine at Mount Sinai
   • Jean-Vianney Haure-Mirande
     Icahn School of Medicine
   • Michelle Ehrlich
     Icahn School of Medicine at Mount Sinai
   • Posted: 27 Jan 2023

   TREM2 and DAP12/TYROBP are microglial proteins that form a complex involved in sensing extracellular debris in the interstitial spaces of the brain. Mutations in both genes are linked to neurodegenerative disorders. Missense mutations in both genes are associated with Alzheimer’s disease (AD), while loss-of-function and deletion mutants are associated with Nasu-Hakola disease (NHD). Here, Zhou et al. describe the transcriptomic profile of human brains from NHD patients who harbored homozygous loss-of-function mutations in DAP12. The microglia in the human NHD brains showed a unique signature of injury-response genes, particularly those in pathways including RUNX1, STAT3, and TGFb. This signature overlapped with others that have been associated with interleukin 10 stimulation, and NicheNet analysis implicated FGF2 as the ligand most likely to be responsible.

   Next, Zhou et al. turned to brains from mice expressing a mutant form of DAP12/TYROBP, known as KDY75. This mouse was created by with the intention of impairing DAP12/TYROBP dimerization and signaling, a goal that succeeded through an unusual strategy in which a polypeptide that does not exist in nature, G75-I90, was substituted for the wild-type dimerization domain Y75-R86 (Tomasello et al., 2000). The spliced-in G75-I90 domain retains one of the two phosphorylatable tyrosine residues present in the wild-type Y75-R86 domain. The inclusion of a nonphysiological, potentially phosphorylatable domain raises the question of how to interpret RNA sequencing data derived from KDY75 mice, particularly relative to more conventional knockout mice. KDY75 mice were impaired in their ability to develop the age-associated microglial (AM) signature that has now become associated with the response to Aβ. The authors conclude that the AM/Aβ response phenotype requires DAP12/TYROBP signaling.

   Our laboratory has been investigating the role of DAP12/TYROBP in microglial signaling in mouse models of AD. Our experience using a conventional, constitutive DAP12/TYROBP knockout, deleting exons 3 and 4, is consistent with Zhou et al.’s report using KDY75 mice: i.e., in the absence of an immune stimulus or pathology, the transcriptomic effects of both knockout strategies are modest (Haure-Mirande et al., 2017, 2019, 2022). We observed that the AM/Aβ response phenotype was reduced when we crossed conventional DAP12/TYROBP knockouts with PSAPP mice. Unexpectedly, however, the DAP12/TYROBP deficiency sustained normal learning/memory and electrophysiology in the face of the identical amyloid burden of PSAPP mice.

   In unpublished work, we have performed single-cell sequencing on the hippocampus from the Tyrobp -/- D exons 3,4 conventional knockout mice (age 20 months). With a focus on microglia, the divergence between top microglial differentially expressed genes (DEGs) ranked by adjusted p value is an obvious metric, demonstrating key differences between the two lines, and few of the top-ranked DEGs in the KD75 model are even significant in the Tyrobp -/- D exons 3/4 line (e.g., fewer than 10 percent are shared at p<0.05 and fewer than 30 percent are shared at p<0.1). In our opinion, the limited overlap between the microglial DEGs from the KDY75 mice and those of the conventional DAP12 knockout argues that the KDY75 DAP12 mouse is not equivalent to a conventional DAP12 knockout. There are also major differences between the two mice in transcriptomic changes in other cell types. However, while it is unclear whether these differences are functionally important, there is a very important similarity in that neither model recapitulates NHD.

   References:

   Haure-Mirande JV, Audrain M, Fanutza T, Kim SH, Klein WL, Glabe C, Readhead B, Dudley JT, Blitzer RD, Wang M, Zhang B, Schadt EE, Gandy S, Ehrlich ME. Deficiency of TYROBP, an adapter protein for TREM2 and CR3 receptors, is neuroprotective in a mouse model of early Alzheimer's pathology. Acta Neuropathol. 2017 Nov;134(5):769-788. Epub 2017 Jun 13 PubMed.

   Haure-Mirande JV, Wang M, Audrain M, Fanutza T, Kim SH, Heja S, Readhead B, Dudley JT, Blitzer RD, Schadt EE, Zhang B, Gandy S, Ehrlich ME. Integrative approach to sporadic Alzheimer's disease: deficiency of TYROBP in cerebral Aβ amyloidosis mouse normalizes clinical phenotype and complement subnetwork molecular pathology without reducing Aβ burden. Mol Psychiatry. 2019 Mar;24(3):431-446. Epub 2018 Oct 3 PubMed.

   Haure-Mirande JV, Audrain M, Ehrlich ME, Gandy S. Microglial TYROBP/DAP12 in Alzheimer's disease: Transduction of physiological and pathological signals across TREM2. Mol Neurodegener. 2022 Aug 24;17(1):55. PubMed.

   Tomasello E, Desmoulins PO, Chemin K, Guia S, Cremer H, Ortaldo J, Love P, Kaiserlian D, Vivier E. Combined natural killer cell and dendritic cell functional deficiency in KARAP/DAP12 loss-of-function mutant mice. Immunity. 2000 Sep;13(3):355-64. PubMed.

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References

News Citations

1. Chimeric Mice: Can They Model Human Microglial Responses? 26 Apr 2019
2. Human Microglia Make Themselves at Home in Mouse Brain 1 Aug 2019
3. Healthy, Drug-Resistant Microglia Reinvigorate Mouse Brain 6 Jan 2023

Paper Citations

1. Paloneva J, Autti T, Raininko R, Partanen J, Salonen O, Puranen M, Hakola P, Haltia M. CNS manifestations of Nasu-Hakola disease: a frontal dementia with bone cysts. Neurology. 2001 Jun 12;56(11):1552-8. PubMed.
2. Paloneva J, Manninen T, Christman G, Hovanes K, Mandelin J, Adolfsson R, Bianchin M, Bird T, Miranda R, Salmaggi A, Tranebjaerg L, Konttinen Y, Peltonen L. Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype. Am J Hum Genet. 2002 Sep;71(3):656-62. Epub 2002 Jun 21 PubMed.
3. Tomasello E, Desmoulins PO, Chemin K, Guia S, Cremer H, Ortaldo J, Love P, Kaiserlian D, Vivier E. Combined natural killer cell and dendritic cell functional deficiency in KARAP/DAP12 loss-of-function mutant mice. Immunity. 2000 Sep;13(3):355-64. PubMed.

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